Platelets from stem cells

Abstract

Human embryonic stem cells are induced to differentiate first into the hematopoietic lineage and then into megakaryocytes, the cells which generate platelets. The proper in vitro culture of megakaryocytes results in the production and shed of platelets. This makes possible, for the first time, the in vitro production of a human blood factor needed by many patients. The in vitro produced platelets lack the immune functions that are the source of incompatibility with current platelet transfusions and thus can be universally transfused to patients without issues of immune rejection.

1 . A method for the production of human platelets in vitro comprising the steps of 
 (a) culturing human embryonic stem cells under conditions which favor the differentiation of the cells into the hematopoietic lineage;    (b) culturing the cells of the hematopoietic lineage into megakaryocytes;    (c) culturing the megakaryocytes so that they will produce platelets; and    (d) recovering the platelets apart from the megakaryocytes.    
   
   
       2 . A method as claimed in  claim 1  wherein the step (a) is performed by encouraging the formation of embryoid bodies and then selectively recovering hematopoietic cells from the embryoid bodies.  
   
   
       3 . A method as claimed in  claim 1  wherein the step (a) is performed by co-culture of the human embryonic stem cells with stromal cells.  
   
   
       4 . A method as claimed in  claim 1  wherein the step (b) is performed by culturing the cells from step (a) in a medium including thrombopoeitin, interleukin  3 , interleukin  6 , interleukin  11 , FLT- 3  and stem cell factor  
   
   
       5 . A method as claimed in  claim 1  wherein the megakaryocytes are positive for CD 41 , CD 42   a , CD 42   b , CD 61 , CD 38 , CD 45  (weak) and CD 62 P, but negative for CD 34 , CD 117 , beta-2-microglobulin, and HLA-DR  
   
   
       6 . A method as claimed in  claim 1  further comprising exposing the culture of megakaryocytes to nitric oxide to encourage the budding of platelets.  
   
   
       7 . A method as claimed in  claim 6  wherein the nitric oxide is produced by adding GMSO to the culture.  
   
   
       8 . A method claimed in  claim 1  wherein the platelets are separated from the megakaryocytes by low speed centrifugation.  
   
   
       9 . Human platelets produced by in vitro culture using the process of  claim 1 .  
   
   
       10 . The human platelets of  claim 9  wherein the platelets are free of immunoglobulin molecules.  
   
   
       11 . Human platelets produced in in vitro culture, the platelets being biologically active to initiate clotting, the platelets being substantially free of and never having been exposed to blood antigens, lymphocytes and constituents of human blood plasma.  
   
   
       12 . An aliquot of human platelets comprising functional human platelets produced in vitro and substantially free of immunoglobulins and substantially free of ABO and Rh antigens.  
   
   
       13 . An aliquot of human platelets produced by in vitro culture, the platelets being biologically active to initiate clotting in vivo, the platelets carried in a medium containing no human serum or plasma.